Non-specific binding of secondary antibodies in IF - Secondaries stain Swiss 3T3 nucleoli (Jan/09/2006 )

Hello all,

I have had difficulties with my secondary antibodies staining the nucleus (faintly), and the nucleoli (medium intensity) in Swiss 3T3 cells. Has anyone observed similar things when doing IF? I'm new to IF, is there something I'm missing?

Antibodies I have tried:
* FITC-conjugated goat IgG (anti-rabbit) from Sigma, affinity purified
* As above but from Chemicom.
* Texas-red conjugated donkey IgG (anti-sheep) from Sigma, affinity purified.

I found that all of these in the absence of primary antibody, stained the nucleus faintly and the nucleolus with medium intensity, giving crisp outlines. No autofluorescence was seen. It was not as strong as the fluorescence seen when I stained with texas-red phalliodin or PI, but it was still quite clear.

I've tried fixing with 2% paraformaldehyde (15 minutes at RT) or methanol (at -20C), permeabilisation with 0.1% SDS or 0.2% triton-X, and I've tried including 0.05% Triton-X in the PBS for the washing steps, but to no avail. I blocked in PBS + 1% BSA (1 hour at 4C), and always wash 5 times, 5 minutes each, in ice-cold PBS between steps.

Any thoughts or suggestions would be greatly appreciated!

i personaly block with 0.5% bsa for 1h in 37C
try it